The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.


Introduction
2][3] Delivering polyubiquitinated proteins to the autophagosome or proteasome, Sequestosome-1/p62 plays a key role in the degradation of aggregate prone proteins. 1STM1 gene mutations may act as a potential threat by causing altered autophagy, resulting in pathogenic protein aggregation and the development of a variety of neurodegenerative diseases, including ALS and FTD. 4 Furthermore, SQSTM1 mutations have been identified in patients with ALS and FTD. 5 Serving as a signalling hub for neurodegenerative pathways, Sequestosome-1/p62 poses as a prospective therapeutic target in the treatment of neurodegenerative diseases.6 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data (1-3).Here we evaluated the performance of seventeen commercial antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation, and immunofluorescence, enabling biochemical and cellular assessment of Sequestosome-1 properties and function.The platform for antibody characterization used to carry out this study was endorsed by a committee of industry academic representatives.It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available antibodies against the corresponding protein.The standardized consensus antibody characterization protocols are openly available on Protocol Exchange, a preprint server (https://doi.org/10.21203/rs.3.pex-2607/v1).7 The authors do not engage in result analysis or offer explicit antibody recommendations.A limitation of this study is the use of universal protocols -any conclusions remain relevant within the confines of the experimental setup and cell line used in this study.Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles.This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Gidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway.8

Results and discussion
0][11] The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal using antibodies.To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million "TPM" + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655).U2OS, expressing the Sequestosome-1 transcript at 6.7 log 2 (TPM+1), was identified as a suitable cell line and was modified with CRISPR/Cas9 to KO the corresponding SQSTM1 gene (Table 1).

REVISED Amendments from Version 1
In this second version of the Data Note, we have included two paragraphs to the end of the Introduction section to clearly define the mission of the YCharOS initiative to the readers while clarifying that they present antibody characterization openly, without restriction, while leaving the result analysis up to the readers.Furthermore, the reference to the standardized antibody characterization platform used to carry out this study is now included, to allow readers to understand or replicate the workflow.Finally, a detailed description of how the cell line was chosen is now provided in the Results and discussion section.
Any further responses from the reviewers can be found at the end of the article For Western blot experiments, we resolved proteins from WT and SQSTM1 KO cell extracts and probed them side-byside with all antibodies in parallel (Figure 1).
For immunoprecipitation experiments, we used the antibodies to immunopurify Sequestosome-1 from U2OS cell extracts.The performance of each antibody was evaluated by detecting the Sequestosome-1 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).
For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).
In conclusion, we have screened Sequestosome-1 commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and characterized several high-quality antibodies under our standardized experimental conditions.The underlying data can be found on Zenodo. 12,13

Antibodies
All Sequestosome-1 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 14Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat.number 65-6120 and 62-6520).Alexa-555-conjugated goat anti-mouse and antirabbit secondary antibodies are from Thermo Fisher Scientific (cat.number A21424 and A21429).

Cell culture
Both U2OS WT and SQSTM1 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 15

Antibody screening by Western blot
Western blots were performed as described in our standard operating procedure. 7U2OS WT and SQSTM1 KO were collected in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor (MilliporeSigma, cat.number P8340).Lysates were sonicated briefly and incubated for 30 min on ice.Lysates were spun at ~110,000 x g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western blot.BLUelf prestained protein ladder (GeneDireX, cat.number PM008-0500) was used.
Western blots were performed with large 4-15% polyacrylamide gels and transferred on nitrocellulose membranes.Proteins on the blots were visualized with Ponceau staining, which is scanned to show them together with individual Western blots.Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat.number 800-095) in TBS with 0.1% Tween 20 (TBST) (Cell Signaling Technology, cat.number 9997).Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST.Membranes were incubated with regular ECL (cat.number 32106) or super signal West Femto (cat.number 34096) from Thermo Fisher Scientific prior to detection with the HyBlot CL autoradiography films from Denville (cat.number 1159T41).Antibody screening by immunoprecipitation Immunoprecipitation was performed as described in our standard operating procedure. 7Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μl of phosphate buffered saline (PBS) (Wisent, cat.number 311-010-CL) with 0.01% triton X-100 (Thermo Fisher Scientific, cat.number BP151-500) in a 1.5 mL microcentrifuge tube, together with 30 μl of protein A-(for rabbit antibodies) or protein G-(for mouse antibodies) Sepharose beads.Tubes were rocked overnight at 4°C followed by several washes to remove unbound antibodies.
U2OS WT were collected in HEPES lysis buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor (MilliporeSigma, cat.number P8340).Lysates were rocked 30 min at 4°C and spun at 110,000 x g for 15 min at 4°C.One ml aliquots at 1.0 mg/ml of lysate were incubated with an antibodybead conjugate for ~2 hours at 4°C.The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of HEPES lysis buffer and processed for SDS-PAGE and Western blot on a 4-15% polyacrylamide gels as described above.

Antibody screening by immunofluorescence
Immunofluorescence was performed as described in our standard operating procedure. 7U2OS WT and SQSTM1 KO were labelled with a green and a deep red fluorescence dye from Abcam (cat.number ab176735 and ab176736), respectively.WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator at 37°C, 5% CO 2 .Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat.number 140770-10ml) in PBS for 15 min at room temperature and then washed three times with PBS.Cells were permeabilized in PBS with 0.1% triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat.number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature.Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary Sequestosome-1 antibodies overnight at 4°C.Cells were then washed 3 x 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/ml for 1 hr at room temperature.Cells were washed 3 x 10 min with IF buffer and once with PBS.Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).
Imaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40x oil objective (NA = 1.40).Analysis was done using ImageJ (RRID:SCR_003070).All cell images represent a single focal plane.Figures were assembled with Adobe Photoshop (version 24.2.1) (RRID:SCR_014199) to adjust contrast and apply 1-pixel Gaussian blur, and then they were assembled with Adobe Illustrator (version 27.3.1)(RRID: SCR_010279).

Guoqiang Xu soochow university, Jiangsu, China
This paper used the WT and Sequestosome-1 knockout U2OS cells to evaluate the quality of the anti-Sequestosome-1 antibodies obtained from different companies in three different applications, i.e.WB, IP, and IF.The results are very useful for other scientists to choose the right antibody for the specific application.
In the paper, WT and Sequestosome-1 knockout U2OS cells were used for the experiment.Additional cell lines, especially for different cancer cell lines and neuronal cell lines should also be tested since the antibodies will be most probably used in these cell lines in real situations.In addition, different cell lines have different levels of protein expression and post-translational modifications, which are also frequently affecting the experimental results.
For Figure 2, why do only two blots have bands for Heavy chains (HC)?An explanation should be provided in the figure legend or the text for better understanding.
For Figure 3, it is recommended that the authors provide the green, red, and contrast images for each antibody for better visualization and assessment.
A conclusion should be provided at the end of the paper to show the quality and application of the different antibodies tested in this paper.A comparison among different antibodies is very valuable for other scientists.In addition, it will be useful to discuss the possible advantages and disadvantages of this work.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: biochemistry, pharmacology, cancer biology, proteomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
might have had.Please see our responses to your specific comments below.
In the paper, WT and Sequestosome-1 knockout U2OS cells were used for the experiment.Additional cell lines, especially for different cancer cell lines and neuronal cell lines should also be tested since the antibodies will be most probably used in these cell lines in real situations.In addition, different cell lines have different levels of protein expression and post-translational modifications, which are also frequently affecting the experimental results.
Response: We understand how the cell line selection process was not well understood, and we have edited the first paragraph of the Results and discussion section to clarify this part of our protocol.From analyzing the public expression database on Depmap, we found that HAP1 expressed PPP2R5D transcript at 6.7 log 2 (TPM+1), which is significantly above the minimum threshold level, 2.5 log 2 (TPM+1), previously established.A limitation of the orthogonal knockout (KO) based approach to test antibodies is that only one cell line (WT and KO) is used to evaluate the performance of the antibodies.However, the strength of our approach lies in the simultaneous testing of multiple antibodies from a variety of commercial sources using WT and an equivalent KO cell line, aimed at identifying the most suitable antibodies for specific applications.A detailed description of the orthogonal strategy applied to select cell lines for the study is outlined in our antibody characterization platform, available on Protocol exchange and (DOI:10.21203/rs.3.pex-2607/v1).This reference has been included in the new version.
For Figure 2, why do only two blots have bands for Heavy chains (HC)?An explanation should be provided in the figure legend or the text for better understanding.
Response: In the protocol exchange paper, previously referred to, which outlines the antibody characterization procedure followed in this article, and is now referenced in the new version of the article.Within the protocol, we have a troubleshooting section with proposed solutions to prevent the heavy chains from appearing.It can be found in the IP Procedure section 2E).Although we have tried to optimize the workflow, we cannot control whether the heavy chain bands appear and show the IP results as we do with all the other antibodies tested.
For Figure 3, it is recommended that the authors provide the green, red, and contrast images for each antibody for better visualization and assessment.
Response: This underlying data is provided in the Dataset, found in the Data availability section.You can also use the following link to be redirected to the Dataset, https://doi.org/10.5281/zenodo.7709902.
A conclusion should be provided at the end of the paper to show the quality and application of the different antibodies tested in this paper.A comparison among different antibodies is very valuable for other scientists.In addition, it will be useful to discuss the possible advantages and disadvantages of this work.
Response: The authors do not participate in results analysis nor offer explicit antibody recommendations, therefore preventing them from identifying which antibodies were highperforming in each respective application.Due to the confines of the experimental setup, the factors that can influence the performance of antibodies and cell line used, we do not draw conclusions.We leave this analysis up to the viewers and within our gateway we feature an editorial that can be used as a guide on how the scientific community can utilize and analyze the YCharOS data to identify high-performing antibodies within the confines of our protocol (DOI: 10.12688/f1000research.141719.1).Furthermore, given that that the article is formatted as a Data Note, it does not require the results to be discussed or concluded.
That being said, we understand how this aspect of the YCharOS initiative was not clearly defined and in the newly submitted version we have included two paragraphs to the end of the introduction that will provide an understanding as to why we do not analyze the results, preventing further misinterpretation.
Competing Interests: No competing interests were disclosed.

Yasukazu Takanezawa
Kitasato University, Tokyo, Japan This paper delves into the intricacies of Western blot, immunoprecipitation, and immunofluorescence techniques utilizing commercially available antibodies targeting p62, particularly in the context of neurodegenerative disease pathogenesis.This paper provides valuable insights into the application of p62 antibodies.Additionally, incorporating co-staining with LC3 would enhance its informativeness.Moreover, exploring the characterization of anti-p62 antibodies from other companies would be advantageous.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes

Reviewer
Report 21 February 2024 https://doi.org/10.5256/f1000research.145562.r247622© 2024 Takanezawa Y.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table 1 .
Summary of the cell lines used.